Apparatus for generating a temperature gradient

ABSTRACT

A novel apparatus for generating temperature gradients is described. The apparatus includes a semiconductive wafer and electrical connectors attached to, preferably, one of the edges of the wafer. Methods for transferring the temperature gradients to strata are described. The temperature gradients on the strata can be used for analyses of molecules, particularly biological macromolecules. The present invention also includes improved methods for determining the thermal stability of binding complexes.

BACKGROUND OF THE INVENTION

This invention relates to an apparatus that generates a thermalgradient, particularly on a wafer. This invention also relates tomethods of using the thermal gradient in molecular interactions,particularly for characterizing interactions involving biologicalmacromolecules.

The stability and interactions of biological macromolecules aredetermined by a number of forces including, for example, ionic forces,van der Waals forces, and hydrogen bonds. Hydrogen bonds are known to befairly weak and heat-labile forces in biological macromolecules. Smallchanges in the environment, in particular the temperature of thebiological macromolecules, can alter the intramolecular and/orintermolecular hydrogen bonding of the macromolecules. Biologicalmacromolecules, thus, can be sensitive to even small fluctuations in theenvironment.

Hybridization between nucleic acid molecules requires successfulformation of hydrogen bonds between complementary nucleic acidmolecules. Because hybridization relies on weak, heat-labile hydrogenbonds, hybridization is an exquisitely temperature sensitive process.Small fluctuations in the temperature and/or in the sequence of thenucleic acids can affect hybridization between complementary nucleicacid molecules.

Currently, information derived from hybridizations conducted ondeoxyribonucleic acid (DNA) chips is stimulating advances in drugdevelopment, gene discovery, gene therapy, gene expression, geneticcounseling and plant biotechnology. A DNA chip is a rigid flat surface,typically glass or silicon, with short chains of related nucleic acidsspotted in rows and columns on it. As an example, hybridization betweena fluorescently labeled single stranded nucleic acid molecule andnucleic acid molecules at specific locations on the chip can be detectedand analyzed by computer-based instrumentation.

Among the technologies for creating DNA chips are photolithography,“on-chip” synthesis, piezoelectric printing and direct printing. Chipdimensions, the number of sites of DNA deposition (sometimes termed“addresses”) per chip and the width of the DNA spot per “address” aredependent upon the technologies employed for deposition. The mostcommonly used technologies presently produce spots with diameter of50-300 micrometers (μm). Photolithography produces spots that can havediameters as small as 1 μm. Technologies for making such chips aredescribed, for example, in U.S. Pat. Nos. 5,925,525 to Fodor et al.,5,919,523 to Sundberg et al., 5,837,832 to Chee et al. and 5,744,305 toFodor et al. which are incorporated herein by reference.

Hybridization to nucleic acids on DNA chips can be monitored, forexample, by fluorescence optics, by radioisotope detection, and massspectrometry. The most widely-used method for detection of hybridizationemploys fluorescence-labeled DNA, and a computerized system featuring aconfocal fluorescence microscope (or an epifluorescence microscope), amovable microscope stage, and DNA detection software. Technicalcharacteristics of these microscope systems are described in U.S. Pat.No. 5,293,563 to Ohta, U.S. Pat. No. 5,459,325 to Hueton et al. and U.S.Pat. No. 5,552,928 to Furuhashi et al. which are incorporated herein byreference. Further descriptions of imaging fluorescently labeledimmobilized biomolecules and analysis of the images are set forth inU.S. Pat. No. 5,874,219 to Rava et al., U.S. Pat. No. 5,871,628 toDabiri et al., U.S. Pat. No. 5,834,758 to Trulson et al., U.S. Pat. No.5,631,734 to Stern et al., U.S. Pat. No. 5,578,832, to Trulson et al.,U.S. Pat. No. 5,552,322 to Nemoto et al. and U.S. Pat. No. 5,556,539 toMita et al. which are incorporated herein by reference.

Currently, manipulations performed with DNA chips are limited toprotocols in which all of the samples on a chip are at about the sametemperature. Simple and inexpensive methods of creating temperaturedifferentials on DNA chips would greatly expand the repertoire ofprocedures available that can be performed on a DNA chip.

SUMMARY OF THE INVENTION

In a first aspect, the invention pertains to an apparatus. The apparatusincludes a semiconducting wafer and two electrical connectors that areadjacent to each other on the wafer. Each of the connectors are attachedto the wafer at an attachment site on the wafer with a gap disposedbetween the two attachment sites. A power source is connected to thewafer through the two electrical connectors.

In a further aspect, the invention pertains to a method of generating atemperature gradient. The method includes attaching two electricalconnectors to a semiconducting wafer, wherein each of the connectors areadjacent to each other and attached to the wafer at an attachment sitewith a gap disposed between the attachment sites. The method alsoincludes connecting a power source to the wafer through the two electricconnectors.

In another aspect, the invention pertains to a method of analyzingbiological macromolecules. The method includes establishing atemperature gradient on a semiconducting wafer having a stratum disposedthereupon. The stratum has one or more samples that include biologicalmacromolecules in thermal contact with the temperature gradient. Thewafer has two electrical connectors connected to opposite poles of anelectrical power source. The method also includes evaluating the samplesto determine thermal stability of complexes formed with the biologicalmacromolecules in the samples. The samples are evaluated by measuring aproperty of the sample.

In a further aspect, the invention pertains to a method of conductingnucleic acid hybridization. The method includes establishing atemperature gradient on a stratum disposed on a semiconducting wafer.One or more samples including nucleic acid molecules are disposed on thestratum that is in thermal contact with the temperature gradient. Twoelectrical connectors are connected to the wafer and to opposite polesof an electrical power source. The method also includes performing ahybridization protocol on the one or more samples to determinetemperature effect based on the gradient.

In yet another aspect, the invention pertains to a method of assessingbinding complex interactions. The method includes establishing atemperature gradient on a semiconducting wafer having a stratum disposedthereupon. The stratum has one or more samples, each sample includingone or more members of a binding complex in thermal contact with thetemperature gradient. The wafer has two electrical connectors connectedto opposite poles of an electrical power source. The method alsoincludes evaluating the samples to determine thermal stability of thebinding complex on the stratum.

In a further aspect, the invention pertains to a method of generating atemperature gradient on a stratum. The method includes placing thestratum in thermal contact on a surface having a temperature gradientwherein the stratum has low thermal conductivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a thermal gradient apparatus.

FIG. 2 is a top view of the thermal gradient apparatus.

FIG. 3 is a side view of the thermal gradient apparatus of FIG. 2.

FIG. 4a is a top view of the wafer of FIG. 2 with three parallel tracksused for temperature measurement.

FIG. 4b is a plot of temperature versus position on the wafer for eachof the tracks indicated on the surface of the wafer in FIG. 4a.

FIG. 5a is a schematic illustration of an apparatus in which atemperature gradient is formed by thermal conduction alone.

FIG. 5b is a plot of temperature versus position for three tracksindicated in FIG. 5a using a silicon wafer in the apparatus.

FIG. 5c is a plot of temperature versus position for three tracksindicated in FIG. 5a using a glass microscope slide in the apparatus.

FIG. 6a is a top view of a thermal gradient apparatus with a glass slideon the wafer.

FIG. 6b is a plot of temperature versus position for each of the tracksindicated on the surface of the slide in FIG. 6a.

FIG. 6c-FIG. 6k are plots of temperature versus position for one of thetracks on the surface of the slide in FIG. 6a when the temperaturecontroller of the apparatus was set to 40° C. 45° C., 50° C., 55° C.,60° C., 65° C., 70° C., 75° C. and 80° C., respectively.

FIG. 7a is a top view of a thermal gradient apparatus with three glassslides on the wafer. Each slide has a drop of water that is covered by acoverslip.

FIG. 7b is a plot of temperature versus position for each of the tracksshown in FIG. 7a.

FIG. 8a is a top view of a thermal gradient apparatus with a fluidiccell on the wafer.

FIG. 8b is a detailed top view of the fluidic cell shown in FIG. 8a.

FIG. 8c is a side view of the fluidic cell shown in FIG. 8b.

FIG. 8d is a plot of temperature versus position for each of the tracksshown in FIG. 8a.

FIG. 9a is a top view of a thermal gradient apparatus with an acrylamidegel formed between two glass slides and placed on the wafer.

FIG. 9b is a side view of the gel assembly shown in FIG. 9a.

FIG. 9c is a plot of temperature versus position for each of the trackson the surface of the slide shown in FIG. 9a.

FIG. 10a is a diagram of a chip containing a single row of immobilizedDNA spots exposed to a 40-70° C. gradient during hybridization with acomplementary labeled nucleic acid probe.

FIG. 10b is a hypothetical result of the experiment depicted in FIG.10a.

FIG. 11a is a diagram of a chip containing three different DNAmolecules, each immobilized in a different row, exposed to a temperaturegradient during hybridization with a labeled nucleic acid probe.

FIG. 11b is a hypothetical result of the experiment depicted in FIG.11a.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been discovered that stable temperature gradients can begenerated on an electrically semiconductive wafer connected to a currentsource. Temperature gradients are generated on the wafer, in a gradientapparatus, by applying a voltage to two physically separated sites onthe wafer. The gradients are stable and approximately linear oversignificant distances.

It has also been discovered that a temperature gradient can betransferred onto or into an adjacent stratum when the stratum is placedin thermal contact with a surface having the temperature gradient, evenif the stratum has low thermal conductivity. These temperature gradientscan be used in efficient approaches for the evaluation of properties ofbiological macromolecules.

The gradient apparatus includes an electrically semiconductive wafer andtwo electrical connectors that are attached to the wafer at adjacentsites, which are referred to as the attachment sites. When the twoelectric connectors are connected to the opposite poles of a powersource, temperature gradients can be produced on the wafer thatgenerally are substantially perpendicular to an attachment line that isderived by connecting the attachment sites. In other words, thetemperature at a given site on the wafer depends on the distance fromthe attachment line.

The wafer can include a proximal edge and a distal edge. The proximaledge of the wafer can include the attachment sites or can be close tothe attachment sites. The side opposite the proximal edge is referred toas the distal edge of the wafer. The attachment line is derived byconnecting the respective edges of the two attachment sites closest tothe distal edge of the wafer. A gradient is formed approximatelyperpendicular to the attachment line. Away from the connectors, thetemperature is approximately independent of displacements along linesparallel to the attachment line. Thus, separate tracks can be definedperpendicular to the attachment line having approximately the sametemperature gradient.

Control circuitry controlling the flow of current between the wafer andthe power source can provide adjustable but stable and reproducibletemperature gradients. The control circuitry can include, for example, atemperature controller, a temperature sensor, a relay switch, atransformer and the like.

Small incremental changes in the temperature can be generated andadvantageously detected on the surface of the wafer. The thermalgradients on the wafer generally are generated by resistive heating ofthe wafer and thermal conduction of the heat. In preferred embodiments,reproducible, stable temperature gradient increments of between about0.1° C./mm and about 1.0° C./mm are generated on the semiconductivewafer. Applications of the stable temperature gradients include, but arenot limited to, determination of the thermal stabilities of duplexnucleic acid molecules, polypeptide: polypeptide complexes, polypeptide:ligand complexes, polypeptide: nucleic acid complexes, polypeptide:lipid complexes, polypeptide: carbohydrate complexes and the like. Asapplied to nucleic acids, the ability to detect thermal sensitivitydifferences can provide diagnostic tools for use in a variety ofapplications.

The temperature gradients generated on surfaces such as a semiconductivewafer can be surprisingly transmitted to one or more stratum placed onthe wafer. The stratum can include, for example, DNA chips, proteinchips, glass microscopic slides, fluidic cells, liquids, acrylamide gelsand the like. When the strata are placed on a wafer in a gradientapparatus of the invention, a thermal gradient can also be detected onthe strata.

Analyses on a variety of biological/chemical molecules can be conductedby using the temperature gradients generated on strata. In particular,thermal stability determinations can be made using the temperaturegradients on a stratum. The molecules can be linked onto or into thestratum. The molecules are generally immobilized on the stratum. By“immobilized”, it is meant that the molecules are not substantiallyremoved or substantially repositioned during subsequent washing or otherexperimental manipulations. Molecules can be immobilized to the stratum,for example, by covalent bonding, hydrophobic bonding, ionic bonding andabsorption.

The biological/chemical molecules can include biological macromolecules,for example, nucleic acid molecules, polypeptides, carbohydrates and thelike. The biological/chemical molecules can also include, for example,drugs, lipids, hormones, ligands and the like, which may or may not bemacromolecules.

Cells and tissues, for example, may also be immobilized on microscopicglass slides by chemically fixing the cells onto the glass slide.Chemical fixatives can include, for example, formalin, ethanol,formaldehyde, paraformaldehyde, glutaraldehyde and the like.

The use of a gradient apparatus of the present invention with DNA chipsor protein chips and various labeled probes has many diagnosticapplications. In one embodiment, temperature-dependent hybridizationsbetween single-stranded nucleic acid molecules immobilized on a chip anda labeled nucleic acid probe can be performed to form a nucleic acidduplex. Nucleic acid molecules at different positions on the chip areexposed to different temperatures based on their location relative tothe temperature gradient. The hybridization signal can be correlatedwith the temperature. The results of these studies can establish therelatedness of similar nucleic acid molecules by estimating the numberof base mismatches between each of nucleic acid molecule samples and thelabeled nucleic acid probe. The gradient apparatus can be used toadvantageously identify even single-base mismatches in a nucleic acidduplex.

In another embodiment, temperature dependent analyses of bindingcomplexes can be performed on chips. The complexes can be, for example,antigen:antibody complexes, enzyme:substrate complexes, receptor:ligandcomplexes, polypeptide: polypeptide complexes, polypeptide:lipidcomplexes, polypeptide:carbohydrate complexes, polypeptide: nucleic acidcomplexes and the like. Binding complexes at different positions on thechip can be exposed to different temperatures based on their locationrelative to the temperature gradient. The results of these experimentscan establish the relative dissociation strengths of related bindingcomplexes.

A. Apparatus

The gradient apparatus of the present invention includes an electricallysemiconductive wafer, a pair of electrical connectors attached to thewafer and a power source. The electrical connectors are adjacent to eachother, but physically separate, when connected to the wafer. Thegradient apparatus may also include control circuitry to obtain adesired temperature gradient.

The semiconductive wafer can be made from a variety of materialsincluding, for example, germanium, silicon, gray (crystalline) tin,selenium, tellurium and boron. The semiconductive wafer is preferablymade from silicon and germanium and more preferably from silicon. Thesemiconductive wafer preferably has a substantially uniform surfacecomposition. The electrical conductivity of semiconductive wafers canbe, for example, between about 10⁴ and about 10⁻³ ohm⁻¹cm⁻¹. Preferably,the electrical conductivity of semiconductive wafers is between about10⁴ and about 10² ohm⁻¹cm⁻¹.

The semiconductive wafer can be doped. Doping can increase or decreasethe conductivity of the wafer. Techniques for doping are known in theart and are described, for example, in Campbell, S. A. The Science andEngineering of Microelectronic Fabrication; New York, Oxford UniversityPress; 1996; pp. 98-102, incorporated herein by reference. Doping agentscan include, for example, boron, phosphorous, arsenic and the like.Preferable semiconductive wafers are boron doped silicon wafers. Dopingof a silicon wafer, for example, can change the electrical conductivityof the silicon wafer from 10⁻¹ ohm⁻¹cm⁻¹ to about 10³ ohm⁻¹cm⁻¹.

The semiconductive wafer is preferably substantially flat and level toretain a stratum placed on the wafer for an indefinite period of time.The wafer, preferably, is also smooth to optimize efficient conductiveheat transfer to the stratum.

In some preferred embodiments, the wafer can include a plurality ofedges, preferably four edges. The wafer may be in the shape of apolygon, for example, a pentagon, a hexagon and the like. The wafer mayalso be curvilinear. The wafer can be substantially shaped, for example,as a rectangle, a square or other parallelograms. The wafer edges mayhave corresponding substantially rectangular edges, square edges and thelike. The corners, for example, may be 90° corners, clipped corners andthe like. Alternatively, the wafer may have rounded corners and thus,can include, for example, ovoid wafers, rectangular-like wafer withrounded corners. Wafers with rounded corners may not have corners thatclearly delineate the different edges. In these embodiments, a rectanglewith corresponding edges can be drawn over the edge of the wafer toapproximate the shape of the wafer. This approximate rectangle can beused to describe the gradient and relative points on the wafer surface.All of these wafers, however, can be used in the gradient apparatusdescribed herein.

Two electrical connectors are generally attached to the wafer adjacentto each other but physically separated. By adjacent, it is meant thatthe two electrical connectors are approximately at the same edge but arenot in physical contact with each other. Preferably, the electricalconnectors are attached to the same edge of the wafer, relative to anapproximating rectangle, if relevant. As described above, the linederived from connecting the attachment site edges, of each attachmentsite, closest to the distal end of wafer is the attachment line.

Generally, a gap is disposed between the attachment sites of theelectrical connectors. The distance between the attachment sites of theelectrical connectors, i.e. the gap, is preferably between about 2 mmand about 180 mm, and more preferably between about 5 mm and about 50mm. The electrical connectors can be edge connectors, for example, cardedge connectors.

The electrical connectors can be connected to the opposite poles of apower source through electrically conductive wires. The power source canbe an alternating current power source or a direct current power source.Preferably, the power source has voltage of between about 2 volts andabout 40 volts and more preferably between about 4 volts and 24 volts.

The temperature gradients on the surface of the wafer are generallysubstantially perpendicular to the attachment line that can be definedas the x-axis. The temperature gradients are, thus, generated along ay-axis, perpendicular to the x-axis (attachment line). In other words,the temperature changes according to the distance from the attachmentline. The temperature is approximately constant at equal distances fromthe attachment line.

Generally, the temperature is highest near the attachment line andprogressively decreases with distance away from the attachment line.Preferably, the decrease in the temperature along any y-axis isapproximately linear. Preferably, movement along the x direction for anyvalue of y does not result in any substantial temperature change.

The temperature along the x direction at or near the attachment line mayvary slightly. The temperatures along a line parallel to and at about 20mm from the attachment line preferably are about the same. Morepreferably, the temperatures along a line parallel to and about 15 mmfrom the attachment line. are about the same. Even more preferably, thetemperatures along a line parallel to and about 10 mm from theattachment line are about the same.

The gradient apparatus preferably includes control circuitry. Thecontrol circuitry of the gradient apparatus can include, for example, atemperature sensor, a temperature controller, a relay switch and atransformer. The temperature sensor is generally physically attached tothe wafer and electrically connected to the temperature controller. Therelay switch and the transformer can be operatively connected betweenthe power source and the wafer. Feedback control between the temperaturesensor and the temperature controller is used to open or close the relayswitch, thereby regulating power to the wafer and controlling itstemperature.

The temperature sensor is generally attached to the wafer surface in thegradient apparatus. The temperature sensor is preferably positioned inthe gap between the two electrical connectors of the gradient apparatus.

The temperature sensor can detect the temperature of the wafer and isassociated with the upper surface of the wafer. Preferably, thetemperature sensor is a resistive temperature sensor, a thermocouple, athermodiode, a thermotransistor, thermoresistor or thermistor. Morepreferably, the temperature sensor is a 100 ohm platinum resistivetemperature detector (RTD). Temperature controllers can be purchasedfrom a number of commercial sources. A suitable temperature controllerincludes, for example, temperature controller model 982 from WatlowEngineering, Winona, Minn.

The temperature controller and the temperature sensor may be parts ofseparately fabricated electrical circuits. Alternatively, they maycomprise a single integrated circuit.

In some embodiments, the gradient apparatus may operate without thefeedback control provided by the temperature controller and sensor. Thegradient can then be dependent on the power source, ambient temperatureand the like.

A variety of suitable relay switches can be used in the gradientapparatus. Suitable relay switches include, for example, a solid staterelay switch, a reed relay switch and the like. Preferably, the relayswitch is a solid state relay switch.

A transformer can be used to change the voltage from a power source, forexample, an alternating current power source, to the wafer. Thetransformer generally is in electrical series with the power source andthe relay switch, for example, as illustrated in FIG. 1.

The apparatus may optionally be mounted in a walled structure thatsupports the wafer in a substantially flat manner and houses thecontroller. The housing may be fabricated from various suitablematerials including plastic and/or metal. Preferably, the housing isplastic, such as polypropylene or polycarbonate so that the housing maybe molded in an inexpensive fashion.

The apparatus may also include a support, preferably molded fromelectrically insulating materials such as silicone rubber, underneaththe distal edge of the wafer.

The apparatus of the invention may also include a commercial personalcomputer, work station or a self-contained microprocessor. In oneembodiment, the computer receives temperature information from thetemperature sensor and executes software commands that cause thecontroller to open or close the relay, thereby regulating the poweravailable to heat the wafer.

In another embodiment, the computer may be electrically attached bytransmission cables to a relay controller and to an electronic sensingdevice (an analog to digital converter) that is electrically connectedto the temperature sensor. In this embodiment, the computer receivestemperature information from the analog to digital converter andexecutes software commands to the relay controller.

FIGS. 1-3 show an illustrated embodiment of a gradient apparatus. Aschematic of gradient apparatus 100 is shown in FIG. 1. Gradientapparatus 100 includes silicon wafer 110, electrical connectors 114 aand 114 b and electrically conductive wires 116 a and 116 b.Electrically conductive wires 116 a and 116 b can be connected to powersource 126 through control circuitry.

Control circuitry can include a number of components. In the embodimentillustrated, control circuitry includes transformer 120, temperaturesensor 130, temperature controller 136 and relay switch 140. Temperaturesensor 130 is positioned in gap 134 between connectors 114 a and 114 b.Temperature sensor 130 and transformer 120 are connected to atemperature controller 136 such as the model 982 available from WatlowEngineering, Winona, MN. A solid state relay switch 140 is connected byelectrical wire 116 b in one electrical series to the controller 136.

A top view of gradient apparatus 100 is shown in FIG. 2. Gradientapparatus 100 includes silicon wafer 110 and two electrical connectors114 a and 114 b attached to wafer 110 at the proximal edge. The distaledge of wafer 110 is supported by wafer gasket 166. Posts 168 supportthe wafer gasket 166 and the electrical connectors 114 a and 114 b.

FIG. 3 is a side view of apparatus 100 shown in FIG. 2. The wafer gasket166 and housing 160 support the distal edge of wafer 110 (the endopposite the electrical connectors 114 a and 114 b).

B. Generation of Temperature Gradients

The gradient apparatus of the present invention can produce stabletemperature gradients on a wafer when the electrical connectors of theapparatus are connected to a power source. The temperature gradientincrements generated on the wafer can be small and reproducible.

The gradient apparatus can be used to establish many differentgradients. In order to generate a desired temperature gradient, thetemperature controller of the apparatus can be set to a desired setpoint temperature. The temperature on the wafer is generally highestnear the attachment line and decreases away from the attachment line.The actual gradient generated on the wafer can depend on, for example,the set point temperature selected, the placement of the temperaturesensor on the wafer, the electrical resistance of the card connectors,ambient temperature and also on the composition of the wafer.

The temperature detected at the attachment line is the upper limit ofthe temperature gradient on the wafer and is preferably within about 20°C., and more preferably, within about 10° C. of the set pointtemperature.

The temperature range of the gradients generated on the wafer can bemanipulated by adjusting the set point temperature of the temperaturecontroller in the apparatus of the invention. The temperature controllercan be set, for example, at about 75° C. to obtain a temperaturegradient of between about 75° C. and about 50° C. Similarly, thetemperature controller can be set, for example, at about 45° C. toobtain a temperature gradient of between about 45° C. and 20° C.

Preferably, the apparatus generates stable temperature gradientincrements of less than about 1° C. per millimeter (mm). Morepreferably, the apparatus generates stable temperature gradientincrements of between about 0.1° C./mm and 0.5° C./mm. The ability togenerate such small temperature gradient increments can be applied tomany different analyses of biological/chemical molecules.

The temperature gradient on the wafer can be determined by measuring thetemperature at various locations along a y-axis. A plot of temperatureversus position, i.e. distance from the x-axis or attachment line, canproduce a temperature gradient profile. The slope of the temperaturegradient profile also can provide the temperature gradient incrementsgenerated on the wafer.

The slope of the temperature gradient profile for a given wafer and forgiven conditions is generally substantially reproducible. The gradientapparatus can be calibrated for a wafer and fixed by setting thecurrent. Once calibrated, reproducible temperature gradients aregenerated by setting the set point of the controller.

The slope of the temperature gradient profile, thus the increments, maybe decreased by additionally attaching a heating source to the wafer atthe distal edge of the wafer. The presence of the connectors asdescribed above and an additional heating source at the distal edge mayresult in a temperature gradient profile with a smaller slope.

The slope of the temperature gradient profile may be increased byattaching a cooling source at the distal end from the connectors. Thecooling can be performed, for example, by using a fan to sweep ambienttemperature across the end opposite the electrical connectors.Preferably, the cooling can be performed by thermoelectric coolingprovided by a peltier device attached to the wafer, an anodized aluminumheat sink attached to the wafer at the distal end and the like.

The temperature gradient generated on the surface of the wafer can bemonitored in a non-obtrusive manner with an infrared-sensitive camerasystem, image acquisition software, data logging software, a memory cardfor storing the acquired data and data plotting software. Other methodsfor monitoring can include methods of gradient analysis involvingplacement of temperature sensitive electronic circuits such asresistance temperature devices in direct contact with the wafer. Thedetectors, however, can act as heat sinks and distort the gradientprofile.

The gradient apparatus of the present invention can generate the desiredtemperature gradients using resistive heating and thermal conduction. Inone embodiment, as shown in FIG. 4a and FIG. 4b, with an ambienttemperature of about 20° C., a temperature gradient of about 0.30° C./mmwas generated on the surface of a boron doped silicon wafer. Inaddition, the plot in FIG. 4b, indicates that the gradient generated canbe monotone and substantially linear. In temperature gradient profilesdescribed herein, the distances are measured from the attachment line,i.e. 0 mm is the attachment line.

For comparison, an apparatus for producing a temperature gradient bythermal conduction alone (no resistive heating) is illustrated in FIG.5a. The thermal conduction apparatus of FIG. 5a includes surface 182that can be either a silicon wafer or a glass microscope slide heated atone end with a 20 ohm resistor. As shown in FIG. 5b, the temperaturegradient when the surface is of a silicon wafer is about 10° C./mm. Themethod of the invention described herein, thus, can produce atemperature gradient that is approximately 30 times shallower than thegradient produced by thermal conduction alone on a wafer.

As shown in FIG. 5c, the gradient produced when the surface is of amicroscope slide can be approximately 3.7° C./mm using thermalconduction alone. The methods described herein, thus, can produce atemperature gradient that is approximately 10 times shallower than thegradient produced by conduction alone on a glass microscope slide.

C. Application of Temperature Gradients

The temperature gradients generated on the semiconductive wafer usingthe gradient apparatus described herein can be transferred to one ormore strata placed on the wafer. The thermal gradients on the stratacan, in turn, be used to assess the thermal stabilities of moleculesincluding, for example, nucleic acids, polypeptides, carbohydrates,lipids, drugs, ligands, combinations thereof and the like. The thermalstability of complexes of the chemical/biological molecules and theirrespective binding partner(s) can be determined using the methodsdescribed herein.

The molecules to be analyzed are generally placed on a stratum such as aglass microscope slide, a silicon chip, an acrylamide gel,nitrocellulose, a charged nylon membrane and the like. By placing thestratum containing the molecules on the wafer of the invention,performing biological manipulations, and then removing the stratum fromthe wafer, the wafer can be reused many times. When biologicalmanipulations are carried out, as shown in the examples below, thetemperature gradient on the wafer is transferred to the stratum.

Samples that can be analyzed in the gradient apparatus can include anynumber of chemical/biological molecules. Samples can include isolated orpurified macromolecule preparations such as isolated nucleic acids andpolypeptides. Samples can also include drugs, hormones, and the like. Inaddition, samples can include tissues, parts of tissues, partiallypurified tissue extractions, cell preparations, living cells and otherbiological material.

Suitable strata can include, for example, glass chips, microscopic glassslides, acrylamide gels, fluidic cells, liquids, coverslips for theglass slides and the like. Strata can be any other strata that areemployed in medical diagnostics, molecular biology and cellular biologyat temperatures, preferably ranging from ambient temperature to about100° C. The thermal conductivity of the strata can vary. Surprisingly,the temperature gradient can be transmitted to strata that have lowthermal conductivity. The temperature gradient can be transferred toglass, for example, that has a thermal conductivity between about0.1-1.0 watts/meter/° K.

Stratum can include one component such as a DNA chip or a protein chip.The stratum can also include a plurality of components, for example, afluidic cell having a base, a glass slide, liquid and a cover, a slideassembly having two slides with acrylamide gel disposed between theslide, and the like.

In particular, a temperature gradient on a wafer of the invention can betransferred to the surface of a glass or silicon DNA chip in thegradient apparatus. Thus, any samples that may be present on the DNAchip would be subjected to the corresponding temperature of the DNA chipat the particular location.

In one illustrated embodiment, glass microscopic slide stratum 190 isplaced on wafer 110 of the invention as shown in FIG. 6a. Thetemperature gradients along the y-axes of the slide were collected andanalyzed by an infrared imaging system and then plotted in FIG. 6b.These results demonstrate that y-axes on microscopic glass slide 190have temperature gradients of approximately 0.3° C./mm.

The temperature gradients along y-axes of the slide when thecontroller's set point was adjusted to 40° C., 45° C., 50° C., 55° C.,60° C., 65° C., 70° C., 75° C. and 80°C., respectively are plotted inFIG. 6c-FIG. 6k. These results demonstrate that the invention canproduce gradients with slopes between 0.1° C./mm and about 0.5° C./mm.The slope of the gradient is determined by the setpoint. Thus, setpointsof 40° C., 70° C. and 80° C. generate, respectively, gradients withslopes of 0.1° C./mm, 0.3° C./mm, and 0.5° C./mm.

In another illustrated embodiment shown in FIG. 7a, apparatus 100includes three glass microscope slides 190 placed on silicon wafer 110.A small drop of water, approximately 50 microliters in volume, can beplaced onto the center of each of the three microscopic slides. Eachdrop of water can then be covered with a glass microscope slidecoverslip 196. The three slide assemblies can then be placed on wafer110 with a preformed temperature gradient and analyzed by thermalimaging after one minute. The plotted results of the apparatus in FIG.7a are shown in FIG. 7b. The results in FIG. 7b indicate that the y-axeson coverslips 196 atop each of the slides 190 can have temperaturegradients of approximately 0.3.0° C./mm.

In another illustrated embodiment shown in FIG. 8a, apparatus 100 with aDNA chip fluidic cell 210 containing glass microscopic slide 190 andliquid film with a volume of approximately 120 microliters is showndisposed on wafer 110. FIG. 8b illustrates a more detailed top view ofthe fluidic cell 210 shown in FIG. 8a. Assembled fluidic cell 210 caninclude base 214, preferably made from lucite, with a machined recess218 that can accommodate microscopic slide 190, a machined groove fittedwith an o-ring 220, lucite lid 240 with two filling holes 224 andmachine screws 230 that tighten the lid against o-ring 220. In use, aglass microscopic slide with DNA is placed in the cell, the lid isplaced on the gasket and the machine screws are used to tighten the lidagainst the gasket. The tightened cell can be water-tight, and has anairspace, approximately 0.1 mm high between the surface of the slide andthe lid, with a volume of approximately 120 microliters. Fluid can beintroduced through one of the fill holes and both fills can then besealed. The assembled cell with a fluid film atop the slide is placed onthe wafer of the invention with a preformed temperature gradient and thetemperature along the lucite lid of the cell is collected and analyzedby an infrared imaging system and then plotted. A plot of thetemperature gradients on the lid of cell is shown in FIG. 8d andindicates that the y-axes of the lid of the cell can have temperaturegradients of approximately 0.3° C./mm.

The temperature gradients can also be transmitted to and through anacrylamide gel disposed between two glass slides. Two glass slides withacrylamide gel between them can be placed on the wafer. In FIG. 9a andFIG. 9b, two glass slides 190 a and 190 b are disposed on the wafer withan acrylamide gel between glass slides 190 a and 190 b. The lineartracks, shown in FIG. 9a, are the tracks on the upper surface of topglass slide 190 b. The temperature on the upper surface of the top glassslide 190 b can be measured using an infrared camera as described above.The upper surface of the top glass slide 190 b can have a temperaturegradient as shown in FIG. 9c. The presence of temperature gradients onthe top glass slide 190 b can indicate that the strata underneath theglass slide 190 b have approximately similar temperature gradients.Thus, the bottom glass slide. 190 a and the acrylamide gel between theglass slides 190 a and 190 b can have approximately similar temperaturegradients as the top glass slide 190 b. The samples in the acrylamidegel can, thus, be exposed to the temperature gradients.

Samples can be placed on the stratum in order to assess thermalstability at a variety of temperatures. Generally, a plurality ofsamples are placed on the stratum at different locations on the stratum.As described above, samples that are placed at differing positions alonga y-axis perpendicular to the attachment line, i.e. samples havingdifferent y-coordinates, are generally exposed to differenttemperatures. Samples that are placed at the same y-coordinates but atdiffering points along an x-axis, i.e. samples have differingx-coordinates but same y-coordinates, are generally exposed to about thesame temperature.

The samples may be placed on the stratum using a variety of techniquesknown in the art. A number of nucleic acid samples, for example, may beimmobilized on DNA chips as described in U.S. Pat. No. 5,925,525 toFodor et al., U.S. Pat. No. 5,919,523 to Sundberg et al., U.S. Pat. No.5,837,832 to Chee et al. and U.S. Pat. No. 5,744,305 to Fodor et al.which are incorporated herein by reference. Nucleic acid molecules, forexample, can also be directly synthesized on the DNA chip by solid phasesynthesis on derivatized chips. The samples may also be immobilized ontothe stratum by chemical crosslinking using crosslinking agents,ultraviolet crosslinking and the like. Crosslinking agents and methodsof crosslinking are known in the art.

The sample placed on the stratum can include a single type of moleculesuch as a preparation of a single stranded nucleic acid molecule, apreparation of an antigen and the like. Potential binding partners ofthese molecules, for example, the complementary strand of the singlestranded nucleic acid, the antibody of a antigen:antibody complex can besubsequently added to the stratum and allowed to bind to the immobilizedsample on the stratum. A labeled probe can be added to the stratum thatdirectly or indirectly binds to the molecules immobilized on thestratum. In some embodiments, the labeled probe is the binding partnerof the immobilized molecule.

The labeled probe can provide a means for detecting the thermalstability of the molecules at the specific location on the stratum.Detection of the thermal stability can be performed by observing,locating and/or quantifying the label that is specifically associatedwith the sample on the stratum. The signal generated for detection ofthermal stability can be derived directly or indirectly from theinteraction between the sample and the labeled probe. Detection and/ormeasurement of the signal of the labeled probe can then be indicative ofand/or correlated with the thermal stability of the molecules on thestratum.

The label of the labeled probe can be, for example, a radioactive atom,a fluorophore, a chromophore, a chemiluminescent reagent, an enzymecapable of generating a colored, fluorescent or chemiluminescentproduct, or a binding moiety capable of reaction with another moleculeor particle which directly carries or catalytically generates thesignal. Binding moieties can be attached to the probe and include, forexample, biotin that binds tightly to streptavidin or avidin anddigoxygenin that is tightly bound by anti-digoxygenin antibodies. Theavidin, streptavidin and antibodies, in turn, are easily attached tochromophores, fluorophores, radioactive atoms, and enzymes capable ofgenerating colored, fluorescent or chemiluminescent signals.

Detection of the labeled probe, bound directly or indirectly to themolecules of the sample, can include detecting fluorescence,chemiluminescence, radiolabels and the like by one or more ofmicroscopy, photography, autoradiography and fluorimetry. Detection canalso be measured, for example, by measuring the visible or ultravioletabsorbance.

In some embodiments, the sample can include a binding complex, i.e. twomembers of a binding complex, such as a double stranded nucleic acidmolecule, a receptor:ligand complex and the like. The binding complexcan be immobilized onto the stratum. When exposed to a temperaturegradient, the binding complex, if dissociated, may exhibit a measurableproperty. For example, a binding complex may have a measurablefluorescence that changes upon dissociation.

The chips generally include a plurality of samples. All of the sampleson the chip may be the same. Alternatively, each track of the chip maycontain different samples. Within each track, the samples are preferablyequivalent so that the equivalent samples can be exposed to differenttemperature. Track, as referred to herein, is a row of samples along oneof the y-axis, i.e. the samples in a track have the same x-coordinateand different y-coordinates.

In the apparatus of the invention, a pair of electrical connectors areattached to the wafer as described above, and connected to a powersource. In preferred embodiments, the temperature controller of thegradient apparatus is set to a desired temperature determined by thenature of the samples to be analyzed.

The samples on the chip are exposed to different temperatures that canbe directly related to position on the stratum due to the gradientprofile of the wafer which is efficiently transferred to the stratum.Generally, the slope of the temperature gradient on the stratum isapproximately similar to the slope of the temperature gradient on thewafer.

The interaction between the labeled probe and the sample or samples atvarious temperatures can be determined since the samples at differentpositions on the stratum are exposed to different temperatures. Analysisof the information generated by these types of studies can identify thethermal dissociation temperature of the specific complexes formed; thatis, the temperature at which the members of the binding complex nolonger remain stably associated with each other because of thermalinstability associated with, for instance, temperature-dependent changesin molecular shape, or temperature-dependent rupture of hydrogen bonds.

In some embodiments, for example, a detectable signal, generated by thelabeled probe and indicative of a nucleic acid hybridization event, maybe present for samples, at locations specifying temperatures of 60° C.and 60.3° C., but not at 60.6° C. The hybridization between the samplesand the labeled probe, thus, is stable at temperatures up to about 60.3°C., but is not stable at temperatures above about 60.6° C.

The apparatus of the invention can be used to determine the thermalstability of nucleic acid duplexes by conducting nucleic acidhybridizations on a stratum on the wafer. Methods for nucleic acidhybridization and thermal dissociation are known in the art. As appliedto DNA chips with the present invention, they would involve thefollowing sequential steps: obtaining or creating a DNA chip containingthe desired array of single stranded nucleic acid molecules immobilizedon its surface; obtaining or creating a suitable labeled nucleic acidprobe; annealing the labeled probe to the immobilized nucleic acids ofthe DNA chip disposed on the wafer having a temperature gradient;removing the unhybridized probe by repeating washing; and detecting theremaining hybridized probe. The labeled probe can be, for example, afluorescently labeled, short single stranded DNA molecule between about12 nucleotides long and about 20 nucleotides long. The results of suchanalyses would provide a graphic representation of thetemperature-dependence of nucleic acid hybridization.

The present invention can include methods of determining the stabilitiesof nucleic acid primers used in polymerase chain reaction (PCR). PCR isa method of amplifying nucleic acid target sequences. PCR is commonlyused, and is described, for example, in PCR, A Practical Approach; M. J.McPherson, P. Quirke, and G. R. Taylor, eds. IRL Press, Oxford, UK,1991.

PCR is a technique involving multiple temperature cycles that result inthe geometric amplification of specific polynucleotides present in atest sample, i.e. target sequences, each time a cycle is completed. Oneof the steps in this amplification of target sequences is thehybridization of a single stranded oligonucleotide referred to as aprimer to a region close to or within the target sequence. Primerspreferably are deoxyribonucleotides. The primers can be between about 12and about 50 nucleotides in length and contain base sequences withWatson-Crick complementarity to sequences on one strand of the targetsequence.

The primers anneal to the target sequence in order for the amplificationto occur. The temperature at which the primer is allowed to anneal withthe target sequence is referred to as the annealing temperature. Adesirable annealing temperature is generally high enough to suppressannealing at non-specific sites but low enough to allow for duplexformation between the complementary primer:target sequences. Atannealing temperatures that are too low, the primer may anneal atnon-specific sites. Thus, it is desirable to identify the highesttemperature at which primer:target sequence duplexes can be maintainedin solution.

In one embodiment of the present invention, the dissociation temperatureof specific PCR target:primer duplexes can be determined. Target DNAscan be placed, for example, in rows along the y-axis of the stratum. Thestratum can then be placed in a fluidic cell, exposed to a temperaturegradient, incubated with labeled primers, and washed. The resultingpattern of labeling can identify the thermal stability of theprimer:target duplex. FIG. 10a and FIG. 10b illustrate an example ofthis embodiment and a hypothetical result. A DNA chip 270 with targetDNA arrayed in a row along the y-axis is shown in FIG. 10a. The targetDNA on the chip is exposed to a gradient of between about 40° C. andabout 70° C., and hybridized with a fluorescent primer. After washing toremove unbound primer, the distribution of label along the y-axis isdetermined. One hypothetical result is illustrated in FIG. 10b. Theresult in FIG. 10b shows that a labeled probe is present at a positionin the stratum corresponding to about 55° C., but is not present at aposition corresponding to about 55.6° C. The optimum temperature forthis primer:target combination, thus, is about 55.0° C.

In another embodiment, the temperature gradient on a wafer can be usedto identify one or more mismatches between related nucleic acidmolecules. In particular, the thermal stability information obtainedfrom performing nucleic acid hybridizations in the gradient apparatuscan be used to identify the percentage of mismatch between two nucleicacid molecules. FIG. 11a illustrates one method of using the gradientapparatus for determining thermal stabilities of closely related nucleicacid molecules. Three samples of DNA differing by one base can beimmobilized on a chip and analyzed. Each of the samples can be placed ona different track with aliquots of the same sample placed at differentpositions within the track. A labeled oligonucleotide probe that isexactly complementary to one of the three DNA samples and covering thearea of the base mismatch(s) in the other two DNA samples can be addedto DNA samples on the chip. The data derived from such a study canresult in data, for example, as shown in FIG. 11b. Using this method asingle mismatch between the labeled probe and the DNA sample can bedetected.

In FIG. 11a and FIG. 11b, DNA chip 280 has three different DNA molecules17 a, 17 b, and 17 c arrayed in parallel rows. Molecule 17 b differsfrom molecule 17 c by one base change, and molecule 17 a differs frommolecule 17 c by two base changes. This DNA chip can be placed in afluidic cell, hybridized to a labeled probe exactly complementary tomolecule 17 c and washed to remove unbound labeled probe. One possibleresult is the distribution of labeled probe along the y-axis for eachsample as shown in FIG. 11b. The highest stability is seen between DNAmolecule 17 c and the probe, followed by DNA molecule 17 b. The probeand DNA molecule 17 a form the least stable complex. This result isconsistent with the fact that there are no mismatches between DNAmolecule 17 c and the probe and that there is one mismatch between DNAmolecule 17 b and the probe. This result is also consistent with thefact that there are two mismatches between DNA molecule 17 a and theprobe. The present invention, thus, can be used to identify single basedifferences between DNA molecules.

The thermal stabilities of various binding complexes can also beevaluated using the gradient apparatus of the present invention. Thebinding complexes may include, but are not limited to, polypeptide:nucleic acid complexes, nucleic acid complexes, polypeptide: polypeptidecomplexes such as antigen: antibody complexes, polypeptide:carbohydrate. complexes, polypeptide: lipid complexes, polypeptide:hormone complexes, receptor: drug complexes and the like. In particular,the thermal stability of antigen: antibody, enzyme: substrate, andreceptor: ligand complexes can be established.

One member of the binding complex can be immobilized on the stratum. Thesecond member of the binding complex is preferably added to the stratumand allowed to bind to the immobilized member to form a binding complex.One of the members of the binding complex may include a label,preferably the second member. Alternatively, a labeled probe may beadded that interacts with only the binding complex and is indicative ofthe presence of a complex.

In some embodiments, binding complexes may be immobilized. The detectionmethods can include signals indicative of either the presence of thebinding complex or dissociation of the binding complex.

In one embodiment, monoclonal antibodies directed against differentepitopes of a single antigen can be immobilized in rows along y-axes ona protein chip. Using a temperature gradient, for example, between about20° C. and about 45° C. and a labeled antigen probe, binding stabilitiescan be determined using procedures similar to the procedures describedfor DNA chips above. Briefly, the labeled antigen probe can be added andallowed sufficient time to bind to the monoclonal antibodies on thechip. Unbound labeled antigen can be washed away. The sites with boundlabeled antigen can be identified using any of the protocols describedabove and correlated with the temperature at the site. The results canprovide thermal stability information related to the various antigen:antibody complexes on the chip.

The strength of interaction between a polypeptide receptor and specifichormones, drugs, and/or other ligands also can be analyzed. Naturalhormones, for example, and their synthetic analogs can be chemicallylinked in rows parallel to the y-axis of a stratum. Using a temperaturegradient, for example, between about 20° C. and about 45° C., thelabeled receptor probe, and the procedures described above, the thermalstabilities of receptor:hormone complexes can be determined.

Candidate drugs for therapeutic use can be chemically linked in rowsparallel to the y-axis of a stratum. Using a temperature gradient, forexample, between about 20° C. and about 45° C., labeled drug receptorprobe, and the procedures described above, the thermal stabilities ofindividual receptor:drug complexes can be assessed.

The discussion described herein relates to some applications of thetemperature gradients generated on a semiconductive wafer. The use ofthermal gradients is not limited to only the applications describedherein. Other applications of using the thermal gradients are alsocontemplated by the inventors and will be apparent to those skilled inthe art.

EXAMPLES Example 1 Generation of a Temperature Gradient on a Wafer

This example illustrates the generation of a temperature gradient on asilicon wafer by resistive heating and thermal conductivity. Thisexample also compares the gradients of the invention with a temperaturegradient formed by thermal conductivity alone.

Temperature gradient formed by resistive heating and thermalconductivity gradient was formed with an apparatus shown in FIGS. 1-3. A114 mm×114 mm×0.6 mm boron doped silicon wafer was diced from a circular150 mm wafer purchased from WaferNet (San Jose, Calif.). Card edgeconnectors were connected to the wafer and to the power source. Thetemperature controller (model 982; Watlow Engineering; Winona, Minn.),was set to about 75° C. The temperature controller determinedtemperature with the electrical signal received from a temperaturesensor that was a 100 ohm platinum RTD (Minco; Fridley, Minn.). Thetemperature controller/temperature sensor maintained the temperature atabout 75° C. by the technique of proportional integrationdifferentiation (PID) looping known in the art. The temperaturedetermined by the controller was within 1.0° C. of the set point withinfive minutes of operation, and stayed at 75° C.±0.5° C. indefinitely.After the set point had been attained, the temperature profile of thewafer's surface was taken with an IR SNAPSHOT digital camera(InfraredSolutions: Plymouth, Minn.).

FIG. 4a shows three parallel tracks on the surface of the wafer,perpendicular to the attachment line drawn between the connectors of theinvention, each separated by approximately 5 mm. The temperature versusposition plot along these tracks, shown in FIG. 4b, has three lines thatwere approximately linear, with slopes of approximately 0.3° C./mm.

A thermal conductivity gradient was formed with an apparatus comparableto the apparatus depicted in FIG. 5a. A 20 ohm resistor was glued to oneend of a 25.4 mm×76.2 mm×0.6 mm silicon wafer. The resistor received 10volts of alternating current from a variable-power transformer, andthereby provided heat to one end of the wafer. One hour after power wasprovided to the resistor, the temperature profile of the surface of thewafer was determined as described above.

FIG. 5a showed three parallel tracks on the surface of the wafer,perpendicular to the resistor, each separated by about 5 mm. Thetemperature versus position plot along these tracks, shown in FIG. 5b,has three approximately exponential curves, each with a slope ofapproximately 10° C./mm.

Comparison of the plots in FIG. 4b and FIG. 5b demonstrated that thegradient apparatus of the invention (resistive heating with thermalconduction) produced a shallow linear gradient extending at least 100 mmfrom the source of power, while thermal conductivity alone produced asteep exponential gradient that essentially ends within 10 mm of thesource of power.

Example 2 Generation of a Gradient on a Glass Slide

This example compares the gradient formed on the surface of a glassslide by resistive heating and thermal conductivity to the gradientformed by conductivity alone.

The temperature gradient formed by resistive heating and thermalconductivity was generated as described in example 1. A glass slidehaving dimensions of about 25 mm×75 mm×1 mm was placed on the wafer asindicated in FIG. 6a. The temperature gradient was visualized asdescribed in example 1.

FIG. 6a shows three parallel tracks on the surface of the glass slide,each separated by about 5 mm. The temperature versus position plot alongthese tracks, shown in FIG. 6b, has three lines that were approximatelylinear, with slopes of approximately 0.3° C./mm.

A gradient with thermal conductivity alone was formed with the apparatusdescribed in example 1 and shown in FIG. 5a. A 20 ohm resistor was gluedto one end of a 25.4 mm×76.2 mm×1.0 mm glass slide. The resistorreceived 10 Volts of alternating current from a variable-powertransformer, and thereby provided heat to one end of the slide. One hourafter power was provided to the resistor, the temperature profile of thesurface of the wafer was determined as described in example 1.

FIG. 5a shows three parallel tracks on the surface of the glass slide,each separated by about 5 mm. The temperature versus position plot alongthese tracks, shown in FIG. 5c, produced three exponential curves, eachwith slopes of approximately 3.7° C./mm.

Comparison of the plots in FIG. 6b and FIG. 5c demonstrated that theapparatus of FIG. 6a produced a shallow linear gradient extending acrossthe entire microscope slide, while thermal conductivity alone produced asteep exponential gradient that essentially ended within 20 mm of thesource of power.

Example 3 Generation of Different Gradients on a Glass Slide

This example demonstrates generation of different gradient ranges on aglass slide.

The temperature gradient produced by resistive heating and thermalconductivity was generated as described in example 2, and thetemperature gradient was visualized as described in example 1. To obtaindifferent temperature gradients, the temperature controller was set to40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80°C., respectively.

FIG. 6a shows three parallel tracks on the surface of the glass slide,each separated by about 5 mm. The temperature versus position plot alongtrack 1 at each of the set point temperatures is shown in FIG. 6c-FIG.6k. These results demonstrate that the invention can produce gradientswith slopes between 0.1° C./mm and 0.5° C./mm. The slope of the gradientis determined by the set point temperature. Thus, set points of 40° C.,70° C., and 80° C. generate, respectively, gradients with slopes of 0.1°C./mm, 0.3° C./mm and 0.5° C./mm.

Example 4 Gradient on a Glass Microscope Coverslip

This example illustrates the generation of temperature gradients on thesurface of glass coverslips placed on a glass microscopic slide with adrop of water. The assembly with the glass slide and the coverslips wereplaced on a wafer having a temperature gradient.

The gradient apparatus as shown in FIG. 7a was used and the temperaturegradient was visualized as described in example 1. A drop of water wasplaced on top of 3 glass slides. A glass microscope coverslip was placedon each drop of water. Each coverslip had dimensions of about 22 mm×50mm×0.1 mm. FIG. 7a shows the apparatus with the microscopic glass slidesand the coverslips. FIG. 7a also shows a parallel track on eachcoverslip that was analyzed as described in Example 1. FIG. 7b is a plotof temperature versus position of the three parallel tracks shown inFIG. 7a. As shown in FIG. 7b, a temperature gradient can be formed andmaintained on the surface of the coverslip. The temperature gradientformed on the surface of the coverslip was about 0.3° C./mm.

Example 5 Generation of a Gradient on the Surface of a Fluidic Cell.

This example illustrates the generation of a gradient on the surface ofa fluidic cell containing a glass microscopic slide with DNA.

The resistive heating and thermal conductivity gradient was generated asin example 1. The fluidic cell illustrated in FIG. 8b was positioned onthe wafer of the invention as illustrated in FIG. 8a. Temperature on thesurface of the top plastic cover of the fluidic cell was visualized asdescribed in example 1.

FIG. 8a shows three parallel tracks on the surface of the fluidic cell,each separated by 5 mm. The temperature versus position plot along thesetracks, shown in FIG. 8c, produced three lines that were essentiallylinear, with slopes of approximately 0.3° C./mm. Therefore, thetemperature gradient of the invention can be transferred successivelythrough the lucite base of the fluidic cell, a glass slide, the fluidfilm covering the glass slide and the lucite lid of the fluidic cell.

Example 6 Generation of a Gradient Along the Length of an Acrylamide Gel

This example illustrates the generation along the length of anacrylamide gel with a gradient apparatus of the present invention.

The resistive heating and thermal conductivity gradient was generated asin example 1. A 5% acrylamide gel was formed between two glass slides asillustrated in FIG. 9b and placed on the wafer of the invention asillustrated in FIG. 9a. Temperature on the surface of the top glasscover of the slide was visualized as described in example 1.

FIG. 9a shows three parallel tracks on the surface of the upper glassslide of the acrylamide gel, each separated by about 5 mm. Thetemperature versus position plot along these tracks, shown in FIG. 9c,produced three lines that were essentially linear, with slopes of about0.3° C./mm. Therefore, the temperature gradient of the invention can betransferred successively through a glass slide, an acrylamide gel andanother glass slide.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. An apparatus comprising: a semiconducting waferhaving a surface for a stratum disposed on the wafer; two electricalconnectors each attached to the wafer at an attachment site with a gapdisposed between the two attachment sites; and a power source connectedto the wafer through the two electrical connectors, wherein the powersource causes current flow through the wafer and a continuoustemperature gradient to be formed on the wafer surface.
 2. The apparatusof claim 1 wherein the semiconducting wafer has a substantiallyrectangular shape with corresponding rectangular edges.
 3. The apparatusof claim 2 wherein both attachment sites are near one of the edges. 4.The apparatus of claim 1 wherein the semiconducting wafer comprises asilicon wafer.
 5. The apparatus of claim 4 wherein the semiconductingwafer comprises a doping agent.
 6. The apparatus of claim 5 wherein thedoping agent is selected from the group consisting of boron, phosphorousand arsenic.
 7. The apparatus of claim 1 wherein the electricalconnectors and the power source are connected by electrical wires. 8.The apparatus of claim 7 further comprising control circuitry betweenthe wafer and the power source.
 9. The apparatus of claim 8 wherein thecontrol circuitry comprises a temperature sensor disposed in the gap andelectrically connected to a temperature controller.
 10. The apparatus ofclaim 9 further comprising feedback control between the temperaturesensor and the temperature controller to maintain the measurement of thetemperature sensor within a selected range, and wherein the feedbackcontrol opens or closes a relay switch.
 11. The apparatus of claim 8wherein the control circuitry further comprises an electricaltransformer connected in series between the power source and theelectrical connectors.
 12. The apparatus of claim 1 further comprisingone or more stratum disposed on the wafer.
 13. The apparatus of claim 12wherein the stratum are selected from a group consisting of a DNA chip,a protein chip, a fluidic cell, a microscopic slide, liquid, coverslip,acrylamide gel and combinations thereof.
 14. The apparatus of claim 12further comprising samples disposed on the stratum.
 15. The apparatus ofclaim 14 wherein the samples comprise molecules selected from the groupconsisting of nucleic acid molecules, polypeptides, carbohydrates,lipids, hormones, drugs and combinations thereof.
 16. The apparatus ofclaim 14 further comprising labeled probes disposed on the stratum. 17.The apparatus of claim 16 wherein the labeled probes are selected fromthe group consisting of fluorescent labeled probes, chemiluminescentlabeled probes and radiolabeled probes.
 18. The apparatus of claim 12further comprising members of a binding complex disposed on the stratum.19. The apparatus of claim 1 wherein the temperature gradient formed onthe wafer is perpendicular to an attachment line derived from connectingthe two attachment sites.
 20. The apparatus of claim 1 wherein the waferhas clipped corners.
 21. The apparatus of claim 1 wherein the twoattachment sites are separated by a distance of between about 2 mm andabout 180 mm.
 22. The apparatus of claim 1 wherein the wafer comprises asubstantially uniform composition.
 23. The apparatus of claim 21 whereinthe two attachment sites are separated by a distance of between about 5mm and about 50 mm.
 24. The apparatus of claim 1 wherein the powersource delivers a voltage of between about 2 volts and about 40 volts.25. The apparatus of claim 24 wherein the power source delivers avoltage of between about 4 volts and about 24 volts.
 26. An apparatuscomprising: a semiconducting wafer having a surface for a stratumdisposed on the wafer; two electrical connectors each attached to thewafer at all attachment site with a gap disposed between the twoattachment sites, wherein when a power source is connected across thetwo electrical connectors current flows through the wafer and acontinuous temperature gradient is formed across the wafer surface. 27.The apparatus of claim 26 wherein the semiconducting wafer has asubstantially rectangular shape with corresponding rectangular edges.28. The apparatus of claim 27 wherein both attachment sites are near oneof the edges.
 29. The apparatus of claim 26 wherein the semiconductingwafer comprises a silicon wafer.
 30. The apparatus of claim 29 whereinthe semiconducting wafer comprises a doping agent.
 31. The apparatus ofclaim 30 wherein the doping agent is selected from the group consistingof boron, phosphorous and arsenic.
 32. The apparatus of claim 26 furthercomprising control circuitry between the wafer and the power source, thecontrol circuitry comprising: a temperature sensor disposed in the gapand electrically connected to a temperature controller; and feedbackcontrol between the temperature sensor and the temperature controller tomaintain the measurement of the temperature sensor within a selectedrange, and wherein the feedback control opens or closes a relay switch.33. The apparatus of claim 26 wherein the stratum is selected from agroup consisting of a DNA chip, a protein chip, a fluidic cell, amicroscopic slide, liquid, coverslip, acrylamide gel and combinationsthereof.
 34. The apparatus of claim 26 wherein the temperature gradientformed on the wafer is perpendicular to an attachment line derived fromconnecting the two attachment sites.
 35. The apparatus of claim 34wherein the two attachment sites are separated by a distance of betweenabout 5 mm and about 50 mm.
 36. The apparatus of claim 26 wherein thepower source delivers a voltage of between about 2 volts and about 40volts.
 37. The apparatus of claim 36 wherein the power source delivers avoltage of between about 4 volts and about 24 volts.
 38. An apparatuscomprising: a semiconducting wafer having a substantially rectangularshape with corresponding rectangular edges and a surface for a stratumdisposed on the wafer; two electrical connectors each attached to thewafer at an attachment site near one of the edges and with a gap ofbetween about 2 mm and 180 mm between the two attachment sites, whereinwhen a power source is connected across the two electrical connectorsand delivers a voltage of between about 2 volts and 40 volts, currentflows through the wafer and a continuous temperature gradient is formedacross the wafer surface.
 39. The apparatus of claim 38 wherein thetemperature gradient formed on the wafer is perpendicular to anattachment line derived from connecting the two attachment sites. 40.The apparatus of claim 38 further comprising control circuitry betweenthe wafer and the power source, the control circuitry comprising: atemperature sensor disposed in the gap and electrically connected to atemperature controller; and feedback control between the temperaturesensor and the temperature controller to maintain the measurement of thetemperature sensor within a selected range, and wherein the feedbackcontrol opens or closes a relay switch.
 41. The apparatus of claim 38wherein the two attachment sites are separated by a distance of betweenabout 5 mm and about 50 mm.
 42. The apparatus of claim 38 wherein thepower source delivers a voltage of between about 4 volts and about 24volts.